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When energy intake is restricted in mammals, there are neuroendocrine adjustments in the secretion of reproductive and metabolic hormones to reallocate energy for vital functions. In the present study, we investigated whether there were differences in the luteinising hormone (LH), growth hormone (GH) and cortisol responses to a 48-h fast in adult gonad-intact male and female rhesus macaques. In both male and female macaques, blood glucose levels were significantly lower in fasted than in control studies, and levels were higher in males than in females. Male rhesus monkeys had significantly lower (P < 0.01) mean serum LH levels after a 48-h fast than under fed conditions and this was attributable primarily to a decrease in the amount of LH released during each secretory episode. In fasted females, serum LH levels were significantly greater (P < 0.05) than during the fed conditions but no differences were found in pulse amplitude or in the number of pulses. Almost twice as many GH pulses were observed in both males and females during fasting but there was no difference in either mean serum GH levels or pulse amplitude between control and fasted studies. A typical diurnal profile in cortisol levels was observed in both sexes and both experimental conditions. Under control conditions, male macaques released less cortisol than females, and although fasting increased mean cortisol levels in both males and females, only the males shown a significant rise over levels observed in control studies. The changes in plasma LH and cortisol levels in fasted rhesus macaques are similar to those observed in humans and suggest that gonadotrophin and corticotrophin secretion are more resistant to short-term energy deprivation in female than in male primates.

The first objective of this study was to investigate whether the inhibitory effect of insulin-induced hypoglycemia (IIH) on luteinizing hormone (LH) secretion was the same in unrestrained adult male rhesus macaques as has been previously reported in restrained female macaques. Since IIH did inhibit pulsatile LH secretion in adult male macaques, and some previous studies have implicated arginine vasopressin (AVP) as a central mediator of this inhibition, the second objective was to investigate whether antagonism of AVP action could reverse the IIH-induced inhibition of LH release in males. Ten adult male rhesus macaques (Macaca mulatta) were studied during 15-h periods (07.00-22.00 h), with blood samples collected every 15-min. There were three experimental groups; controls (n=5), IIH (n=6) and IIH plus vasopressin antagonist (AVPa; n=6). During the hypoglycemia studies, the first 5 h served as a control for that occasion and an insulin bolus of 1 U/kg was administered intravenously at 12.00 h. During the IIH plus AVPa, the vasopressin antagonist was infused intravenously from 12.00 h to 17.00 h. LH and testosterone decreased progressively after the insulin bolus in the IIH group reaching a minimum value at 4 h after the infusion. However, compared to the preinfusion levels, secretion of LH and testosterone was not suppressed by hypoglycemia in the group treated with the AVP antagonist. The present study shows that in male macaques not subjected to the psychological stress of restraint, IIH suppresses LH and testosterone secretion. This inhibition of LH release can be blocked in some animals by antagonism of central vasopressin receptors, suggesting that vasopressin is involved in the suppression of gonadotropin releasing hormone/LH release induced by hypoglycemia.

Fasting inhibits the gonadotropic axis and stimulates the corticotropic and somatotropic axes. Since leptin is a product of fat cells that has been implicated in the control of both reproduction and metabolism, we hypothesized that the decrease in leptin observed during fasting was responsible for these effects on reproductive and metabolic hormones. Recombinant rhesus leptin (rrhLep) produced in our laboratory was infused (100 microgram/h) into fasted adult male rhesus macaques (6-9 kg) beginning at midnight after the first missed meal and continuing until the end of the study. Bioactive luteinizing hormone (LH), testosterone, cortisol and growth hormone (GH) were measured in plasma from samples collected at 15-min intervals for the last 15 h (42-57 h) of the fast. We analyzed pulsatile LH and GH secretion by deconvolution analysis and the orderliness of pulsatile LH and GH release by the approximate entropy (ApEn) statistic. There was no difference in LH pulse frequency between control and fasted groups, but there was a significant decrease in the mean concentration of LH released (7.6 +/- 1.4 ng/ml control vs. 2.7 +/- 0.65 ng/ml fasted) that was not relieved with rrhLep infusions (2.8 +/- 0.83 ng/ml). Model-free Cluster analysis confirmed these inferences and also indicated that the peak height was lower in the fasted (4.6 +/- 1.0 ng/ml) and the fasted + rrhLep (2.85 +/- 1.0 ng/ml) groups compared to controls (16. 3 +/- 1.4 ng/ml). Testosterone levels reflected those of LH. Fasting resulted in an increase in GH secretory pulse frequency (5.3 +/- 0. 95 pulses/15 h control vs. 12.8 +/- 1.4 pulses/15 h fasted) and this increase was not affected by rrhLep infusion (12.5 +/- 1.4 pulses/15 h). In addition, fasting also increased the ApEn (decreased the orderliness) of pulsatile GH secretion, and this characteristic was not relieved with rrhLep infusions. Cortisol levels in fasted animals were 2- to 3-fold higher than those observed in control studies, and this increase was particularly pronounced at the time when the animals expected their first meal of the day. The increase in circulating cortisol observed in fasted animals was not affected by rrhLep infusion. Glucose levels at the end of the sampling period were 80 mg/dl in controls, 48 mg/dl in fasted animals and 58 mg/dl in the fasted + rrhLep group. Circulating leptin levels averaged 1.2 +/- 0.37 ng/ml in control animals, 0.7 +/- 0.2 ng/ml in fasted animals and 10.1 +/- 5.6 ng/ml in fasted animals infused with rrhLep. These studies suggest that intravenous replacement with homologous leptin does not reverse the acute changes in GH, LH and cortisol secretion observed with fasting in the adult male macaque.

OBJECTIVE: Because glucocorticoids stimulate leptin release and, at least in vitro, leptin inhibits cortisol secretion, a feedback system between glucocorticoids and leptin has been proposed. However, in humans and non-human primates there are no in vivo studies to support any role for leptin in the control of the hypothalamic-pituitary-adrenal axis. In this study, we investigated the effect of leptin on (i) ACTH-stimulated secretion of cortisol in six male rhesus monkeys and (ii) basal and forskolin (FSK)-stimulated cortisol secretion by the human adrenal carcinoma cell H295R in vitro. DESIGN AND METHODS: In vivo studies: after suppression of endogenous ACTH with either dexamethasone (n=6) or a corticotropin-releasing factor (CRF) antagonist (d-Phe CRF(12-41)) (n=3), 1 microg bolus of human ACTH(1-24) was administered to stimulate adrenal cortisol release. Blood samples were collected every 15 min for 3 h. Leptin (1 mg) was infused over 4 h, starting 1 h before ACTH bolus. In vitro studies: NCI-H295R cells were incubated for 6, 12, 24 and 48 h in the absence or presence of 20 micromol/l FSK in combination with leptin (100 ng/ml medium). Cortisol levels in serum and medium were measured by solid phase radioimmunoassay. RESULTS: Acute leptin infusion to rhesus monkeys did not change basal cortisol levels, peak cortisol levels after ACTH(1-24) or the area under the curve when compared with studies in which leptin was not given. FSK increased cortisol levels in medium at 24 and 48 h, but leptin did not change cortisol release in either control or FSK-stimulated cells. CONCLUSIONS: Short-term leptin infusion affected neither the cortisol response to ACTH in non-human primates in vivo nor cortisol release (basal or FSK stimulated) by H295R cells, in vitro. These data suggest that leptin may not be an acute regulator of primate adrenal cortisol secretion.

OBJECTIVES Although the adipocyte protein leptin has been implicated in the control of reproductive function in rodents, its role in primate reproductive physiology is poorly understood. Because primates in puberty show nighttime LH secretion and there is considerable evidence that the fertile state requires adequate nutrition, we reasoned that animals on the verge of reproductive competence would respond to leptin infusions by secreting LH. Food restriction reduces circulating leptin levels and slows or stops the GnRH pulse generator. Therefore, we examined the endocrine effects of leptin infusions in food-restricted male pubertal primates during the night when they normally secrete LH. In addition, we investigated the effect of leptin on in vitro testosterone production by Leydig cells. SUBJECTS Four pubertal male rhesus macaques (Macaca mulatta), 4–5.5 kg in weight (2.5–4-year-old) were examined in this study. Leydig cells from adult male rats were to investigate in vitro effects of leptin. DESIGN To document that animals had entered puberty, blood samples were collected from each of the four animals at 15-minute intervals for 15 h both during the day and at night. Since at this age animals secrete LH mainly at night, blood samples were collected at 15-minute intervals from each of the four animals on two separate occasions for 15 h between 1500 and 0600h. During the experiment, animals were feeding from 0800 to 0830h, cages were completely cleaned of food at 0900h and the afternoon meal was not given to individual animals on the day they were studied. One of the studies served as the control (food restricted group) and during the other, 2 mg (n = 4) or 0.3 mg (n = 3) of recombinant human leptin was administered intravenously during 2000–0100h (food restricted plus leptin group). Blood samples (1 ml) were collected through the indwelling catheter and immediately transferred from the plastic syringe into chilled glass tubes containing 10 μl 14% EDTA. The samples were centrifuged at 5-h intervals and the plasma withdrawn and stored frozen at − 20°C in polypropylene vials until assays were performed. MEASUREMENTS Bioactive LH was determined and testosterone, cortisol and leptin were measured by radioimmunoassay. RESULTS During daytime experiments in these animals, LH pulses were sometimes observed late in the day and generally continued for 12–15 h. Food-restricted pubertal males showed delayed or absent LH pulses. Short-term leptin administration to food-restricted male rhesus macaques had no effect on LH, testosterone, or cortisol levels either during or after the infusion. Leptin also had no direct effect on basal or LH-stimulated testosterone production in Leydig cells. CONCLUSIONS Our data support the notion that leptin is not the missing signal for the acute suppression of reproductive hormones secretion in food-restricted primates.

Measures of performance, mortality, adrenal weights, plasma glucocorticoid concentration, and selected immune measures were collected in an attempt to define space needs of laboratory mice. Six replications of 3 C57BL/6 male mice per cage were examined while housed on bedding at 5, 10, 15, or 20 in(2) (32.2, 64.5, 96.8, or 129 cm(2)) per mouse. Body weights were not influenced by treatment; however, mice in smaller spaces (5 in(2) per mouse) consumed or wasted more feed and water than mice given greater space allowances. Mice given the least amount of space (5 in(2) per mouse) had greater lymphocyte proliferation in response to the T-cell mitogen PHA than mice given more space. Mice provided 10 in(2) per mouse had greater natural killer cytotoxicity than mice given greater or less space. Mouse mortality was greater as more space was provided. In contrast, adrenal weights and plasma glucocorticoid concentrations were progressively greater with lower space allowances. The National Research Council 1996 recommendation of 15 in(2) per mouse, for this strain and sex of mice, would result in greater mortality and reduced activity of some immune measures. Socially housed male C57BL/6 mice will benefit from less space than recommended by the National Research Council in 1996.

We have previously demonstrated that corticotropin-releasing hormone (CRH) treatment of MA-10 mouse Leydig tumor cells results in a dose-dependent stimulation of progesterone production. In view of this observation we wished to determine the effects of CRH on the synthesis of the steroidogenic acute regulatory (StAR) protein in these cells. StAR is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage enzyme. Treatment of MA-10 cells with the cAMP analogue dibutyryl cAMP (dbcAMP) resulted in a dose- and time-dependent increase in the levels of StAR protein that reached a maximum at 800 microM dbcAMP and within a time period of 6 h. Further, treatment of MA-10 cells with CRH also resulted in a dose-dependent increase in the synthesis of the StAR protein with a maximal response observed at 1 microM. Slightly different from that observed with dbcAMP, the maximal response to 1 microM CRH was seen at 4 h following stimulation. These results indicate that the observed increase in steroid production in response to CRH in MA-10 Leydig tumor cells is similar to that previously seen with trophic hormone stimulation acting through the cAMP second messenger pathway, and that it occurs as a result of an increase in the synthesis of the StAR protein.

Regular menstrual cycles are the result of predictable cyclic changes in reproductive hormones produced by the pituitary and ovary. A few neurons at the base of the brain in the hypothalamus provide the ongoing stimulus, GnRH pulses, for the synthesis and secretion of LH and FSH which, in turn, stimulate the ovaries to produce the steroids estrogen and progesterone and the small peptides inhibin and activin. It is the feedback relationships between the pituitary and ovarian hormones that provide the basis for the cyclic function of the reproductive system. If fertilization occurs, there is the possibility of a pregnancy which will suppress menstrual cycles for the duration of the pregnancy. Nursing or lactation can also suppress the ovarian cycles, but should not be relied on for birth control. The negative feedback relationship between the ovarian steroids and pituitary gonadotropins is the basis by which hormonal birth control works. Synthetic steroid hormones that can be administered orally suppress the secretion of LH and FSH and thus interrupt the growth and development of ovarian follicles.

Relevant for female athletes in general and especially in adolescent female athletes are the long-term consequences of insufficient energy intake that results in menstrual cycle disturbances and low bone mineral density. Prolonged exercise as practiced by elite athletes can lead to serious, long-term health problems if diet is not adjusted to compensate for energy expenditure. A good barometer of how energy intake matches energy expenditure in female athletes is menstrual function. Eumenorrhea is the term used to describe normal length, regular menstrual cycles. Oligomenorrhea refers to infrequent menses or menstrual cycles that occur inconsistently at intervals of 39–90 days. Amenorrhea is the absence of menstrual cycles in a nonpregnant, sexually mature woman. When menstrual cycles become disordered as a result of prolonged or excessive exercise, there very well may be an issue related to insufficient energy intake, and consultation of a health care provider knowledgeable in sports medicine is strongly advised to correct the situation. The standard curriculum for high school and college coaches of female athletes should include the health consequences of exercise-induced menstrual function and specific methods of prevention and treatment.

The actions of corticotropin-releasing hormone (CRH) on steroidogenesis in enriched preparations of mouse and rat Leydig cells were investigated. Primary cultures of purified Leydig cells as well as a Leydig tumor cell line were used in these studies. CRH had a stimulatory effect on steroid production in both isolated preparations of mouse Leydig cells (80-90% Leydig cells) and MA-10 cells (a mouse Leydig tumor cell line). In primary cultures of mouse Leydig cells, CRH was effective over a range of 1 nM-100 nM, while MA-10 cells were responsive over a wider range (10 nM-100 microM). When a submaximal dosage of CRH was given together with a maximal dosage of hCG, steroid production was stimulated even more highly in MA-10 cells. However, when primary cultures of mouse Leydig cells were treated with CRH and hCG, no similar response was observed. In addition, a CRH antagonist, alpha-helical CRH9-41, reversed the CRH stimulatory effect on steroidogenesis in both mouse Leydig cells and MA-10 cells. The accumulation of intracellular cAMP after CRH treatment was dose-responsive to CRH in both cell types, a finding similar to the results described above for steroid production. CRH had no effect on steroidogenesis in rat Leydig cells (60-80% Leydig cells) in the present study. These results indicate that mouse Leydig cells respond to CRH through specific receptors with increased production of cAMP and steroids.

It is generally accepted that corticotropin-releasing hormone (CRH) is the central mediator of stress-activated changes in the pituitary-adrenal axis because it results in the release of ACTH and ultimately increases the systemic levels of cortisol. And, because in some situations CRH also inhibits the hypothalamic release of GnRH, it has been presumed that it plays the central role in stress-related reduction in pituitary-gonadal function as well. We previously have shown that 6 h of restraint stress in intact female rhesus macaques suppresses plasma levels of LH in the follicular but not the luteal phase of the menstrual cycle and that this effect lasts beyond the period of restraint. Since CRH inhibits both the GnRH pulse generator and LH release in ovariectomized macaques and is generally thought to be the central mediator of stress-induced inhibition of gonadotropin release, we investigated the influence of CRH administration on LH in undisturbed intact female rhesus macaques. Blood samples were collected at 15-min intervals for 15 h from a remote site from female macaques in both the follicular and luteal phase. During this time, each animal received a 4-h infusion of CRH (100-Lg bolus followed by 100 pg/h for 4 h) through an indwelling jugular catheter. Blood samples were collected for an additional 8 h after cessation of the CRH infusion. Cortisol levels were significantly elevated during and after the CRH infusion and were comparable to levels observed in animals that experienced restraint. However, CRH did not suppress LH levels in either the follicular or the luteal phase. These data suggest that CRH is not the only mediator of stress-induced inhibition of gonadotropin release in primates.

The present study investigated the question of how restraint affects the hypothalamo-pituitary adrenocortical axis and the hypothalamo-pituitary gonadal axis in intact, adult female rhesus macaques in both the follicular and luteal phases of the menstrual cycle. Restraint was chosen because it is not physically harmful to the animal but rather serves primarily as a psychological stressor. Blood samples were collected from a remote site at 15-min intervals beginning at 0700 h from tethered adult female rhesus macaques. Each animal was subjected to 6 h of chair restraint after a 3-h control period in the animal's home cage. Samples were collected for an additional 6 h after the end of the restraint period, when each animal was returned to its home cage. Brief anesthesia with ketamine (administered through indwelling catheter) facilitated transfer of the animals to and from the chair. Blood samples were also collected from undisturbed females in both the follicular and luteal phases to document LH, cortisol, and progesterone secretion throughout the day. Plasma ACTH and cortisol, measured as indices of stress, were elevated within 15 min after initiation of restraint and remained elevated after the animals were returned to their cages. In animals sampled in the follicular phase, mean plasma LH levels were lower during restraint and remained suppressed for several hours after the animals were removed from restraint. LH levels were not significantly inhibited by restraint in the luteal phase. When the opiate receptor antagonist naloxone (Nx; 5 mg bolus plus 5 mg/h) was given beginning 2 h after the initiation of restraint, LH levels were elevated compared to prerestraint levels in both phases of the menstrual cycle. These data indicate that restraint is a potent activator of the pituitary-adrenal axis and that at least in the follicular phase of the menstrual cycle, restraint inhibits pituitary LH release. This inhibition of gonadotropin release may involve endogenous opiate suppression of GnRH release, since Nx reversed the effect of restraint.

We previously have shown that 6 h of restraint stress in intact male rhesus macaques (Macaca mulatta) suppresses plasma levels of both LH and testosterone and that this effect lasts beyond the period of restraint. Since corticotropin-releasing hormone (CRH) inhibits both the GnRH pulse generator and LH release in ovariectomized macaques and is generally thought to be the central mediator of stress-induced inhibition of gonadotropin release, we investigated the influence of CRH administration on LH and testosterone in unrestrained intact male rhesus macaques. Blood samples were collected from 5 intact male macaques at 15-min intervals for 15 h from a remote site. During this time, each animal received a 4-h infusion of CRH (100-micrograms bolus followed by 100 micrograms/h for 4 h) through an indwelling jugular catheter. Blood samples were collected for an additional 8 h after cessation of the CRH infusion. ACTH and cortisol levels were significantly elevated during and after the CRH infusion and were comparable to levels observed during restraint. Although LH levels appeared lower in animals given CRH, they were not different from those in untreated control males. In some animals, CRH appeared to stimulate or prolong LH release. Testosterone levels in CRH-treated animals were significantly lower than in controls both during and after CRH administration. In some instances, increases in plasma LH were not accompanied by a rise in testosterone. This would suggest that as was observed in restrained animals, stress exerts either a direct or an indirect inhibition of testicular testosterone secretion. The present data indicate that this effect may be mediated by CRH.

Crossbred pigs were used to evaluate the effects of shipping stress on natural killer (NK) cell activity, leukocyte numbers, plasma cortisol, and BW changes. In the first study, pigs were bled at a commercial farm and, after shipping, resident and shipped pigs were bled again. Plasma cortisol concen- trations were not different (P > .lo) because of large variation in cortisol concentrations. Furthermore, NK cytotoxicity was nondetectable among all pigs. A second study showed that plasma cortisol concentra- tion rose by approximately 2.6 ng/mL (P = .018) for each minute after pigs were aroused. In the third, more controlled study, pigs were housed in pens of three pigs each. Video recordings were made during the first 24 h pigs were grouped to identify socially dominant, intermediate, and submissive pigs. At time zero (before shipping), resident pigs and those to be shipped had similar plasma cortisol concentrations. However, after the 4-h shipping experience, shipped pigs had elevated (P < .05) plasma cortisol compared with resident control pigs. Shipped pigs lost 5.1% of their BW (P < .05) compared with resident pigs, which gained .02% of their BW. Body weight change during shipping and plasma cortisol were negatively

Three experiments were performed to determine the effect of stress on the neuroendocrine-immune system in nonhuman primates. In Experiment 1 the diurnal variation in cell and hormone levels was determined. The percentages of neutrophils, monocytes, and eosinophils fluctuated throughout the 24-hr period, while white blood cell (WBC), neutrophil-to-lymphocyte ratio (N:L), hemoglobin (Hgb), natural killer cell cytotoxicity (NK activity) and beta-endorphin levels did not. Experiment 2 investigated the effects of ketamine and restraint on behavior. Scratching was increased in control monkeys and animals receiving ketamine, whereas passivity was increased in chair-restrained animals. In Experiment 3, eight adult male rhesus monkeys were restrained in primate chairs at 0600h. Behavior was filmed for 3 hr and blood samples were collected at 0700, 0800, and 0900. Whole blood was analyzed for total WBC and percentage of each leukocyte type. NK activity was also measured. Plasma levels of cortisol and beta-endorphin were determined and behavior was quantitated from video-records. WBC and the percentage of neutrophils increased during the restraint period, while the percent lymphocytes and monocytes decreased. NK activity also decreased over time after restraint whereas plasma cortisol and beta-endorphin levels increased significantly. Although after the 3 hr of restraint stress, changes were found in hormone levels, behavior, and NK activity, there were no significant correlations between the parameters measured. Thus, our results indicate that there is not a common neuroendocrine response or single neuroendocrine mediator that results in predictable behavioral changes and immune suppression following stress.

Central release of CRH has recently been implicated in modulating natural killer cell (NK) activity independent of its role in activation of the hypothalamic-pituitary-adrenal axis. In the present study, NK- and interleukin-2 (IL-2)-activated NK cytotoxicity against K-562 target cells was examined in porcine lymphocytes cultured in vitro with ACTH or pig lymphocytes after iv or im ACTH administration. Physiological concentrations of porcine ACTH (10(-8)-10(-11) M) added to the culture medium had no direct influence on NK- or IL-2-stimulated NK cytotoxicity. In a second experiment four unrestrained pigs with indwelling catheters given an iv bolus of vehicle or ACTH (1 IU/kg BW) at 0800 h showed significantly elevated cortisol levels for 3 h after ACTH. Although serum cortisol had returned to baseline by 4 h after ACTH treatment, NK- and IL-2-stimulated NK cytotoxicity was dramatically elevated (P less than 0.01) compared to that in saline-injected controls. NK cytotoxicity in control pigs followed a diurnal pattern, with low morning and high evening cytotoxicity. Exogenous ACTH, given by bolus in the morning, prevented the normal morning decline in NK cytotoxicity. Because of this unexpected dramatic increase in NK- and IL-2-stimulated NK cytotoxicity in animals given ACTH, the experiment was replicated in two subsequent studies using 16 pigs (8 controls and 8 experimental) in each. Pigs were injected im with either ACTH (1 IU/kg BW) or an equivalent volume of saline at 0600 h. Two hours later, blood was collected by venipuncture to determine NK cytotoxicity and measure the cortisol response. As was observed in the previous study, NK- and IL-2-stimulated NK cytotoxicity was significantly greater (P less than 0.01) than that in saline-injected controls. In the final experiment pigs were given either ACTH (1 IU/kg BW) or an equivalent volume of saline at 1800 h. Two hours later, blood was collected by venipuncture to determine NK cytotoxicity and cortisol response. ACTH administered in the evening increased NK cytotoxicity, but the effect was only marginally significant and far less dramatic than in previous studies. Because ACTH had little effect on NK- and IL-2-stimulated NK activity in vitro, we hypothesize that the stimulatory effect of exogenous ACTH is mediated through an indirect mechanism, possibly through the suppression of central CRH as a result of elevated cortisol. This effect is more pronounced when the stimulatory dose of ACTH is given at a time in the circadian cycle when NK cytotoxicity is normally low.

The association of fertility with fatness is an ancient concept. Artifacts from France and Austria thought to be at least twenty thousand years old and considered to be representations of fertility goddesses (Schneider and Wade, 2000) depict women who would be considered obese by today standards. Although generous stores of body fat in women is associated with fertility, obese mice (Zhang et al., 1994) and humans (Montague et al., 1997; Strobel et al., 1998) who suffer from a mutation in the gene that codes for either the adipocyte hormone leptin, or for its receptor, are infertile. This suggests that a circulating signal from fat might be important for both appetite control and for fertility.

Leptin is a circulating protein produced by adipocytes that has been implicated in control of body weight through appetite regulation and control of reproduction, most likely through an effect on the central nervous system. From studies in mice, it is clear that the genetic background of the animal on which the mutation in the leptin gene is placed can influence how that mutation is expressed. Although the effects of leptin have been more thoroughly documented in nonprimate species than in primates, a few human families with genetic mutations of the gene for leptin or the leptin receptor show obesity and impaired fertility. There is conflicting evidence regarding the effects of leptin in male primates, but it appears that the metabolic and reproductive effects of low leptin levels caused by reduced energy intake in adult animals can be more readily alleviated by administration of energy substrates than by administration of leptin.

In male mammals, the neuroendocrine responses to fasting include increased GH and cortisol secretion and suppressed LH and T levels. Because blood glucose levels fall during fasting, we hypothesized that this modest, but consistent, change in blood glucose was a metabolic signal for the neuroendocrine adjustments of reproductive and metabolic hormones. Glucose (d-dextrose, 480 kcal/d) was infused into fasted (48 h) adult male rhesus macaques; and LH, cortisol, and GH were measured in plasma from samples collected at 15-min intervals for the last 15 h of the fast. We analyzed hormone secretion by deconvolution analysis, and the orderliness of release patterns by the approximate entropy statistic. Circulating blood glucose was 76 ± 7 mg/dl in the fed control group, significantly higher (P < 0.01) than the level of 56 ± 3 mg/dl in the fasted group. The increase in GH pulsatility and the 2-fold elevation in cortisol levels observed in the fasted male macaques were prevented by parenteral glucose delivery. The suppression of LH in fasted animals was not relieved by glucose infusions but seemed to be partially prevented in three of the animals. These findings are consistent with the hypothesis that glucose serves as a signal of nutritional status controlling adaptive neuroendocrine responses to fasting in the primate.

During puberty, primates begin to secrete LH, and presumably GnRH, at night and then eventually throughout the day as they mature. We examined the role of vasopressin, a putative inhibitor of the GnRH pulse generator, on LH secretion in pubertal male macaques both during the day when the GnRH pulse generator was not active and during the night when pulsatile LH secretion was observed. As has been found in other primates, LH and testosterone levels were low during the day and elevated at night. A potent vasopressin receptor antagonist (VPa) was administered during the middle 5 h of a 15-hour daytime or nighttime blood collection period to determine the effects on LH, testosterone and cortisol secretion. Both during the day and night, cortisol secretion was elevated during VPa infusion, suggesting that this V1a receptor antagonist has agonist activity on the V1b receptor in the pituitary involved in vasopressin-stimulated corticotropin release. Mean LH levels during VPa infusion were not different from the control period for that group during the day when LH secretion was absent. Mean LH levels were significantly higher (p < 0.05) than pre-VPa LH levels at night when pulsatile LH secretion was observed. However, LH levels at night during VPa were not higher than levels in untreated animals at the same time. The results of these studies demonstrate that LH and testosterone profiles are similar to those observed in human males during the pubertal transition and suggest that the absence of daytime gonadotropin release during puberty is not due to inhibitory vasopressinergic tone on the GnRH pulse generator.

Our recent observations (1) that there is a difference in circadian patterns of plasma cortisol levels between male and female macaques and (2) that after gonadectomy these differences in the patterns and in the levels of cortisol were reduced prompted us to investigate how 17 beta-estradiol (E2) and progesterone affect cortisol secretion in orchidectomized male rhesus macaques. Five male macaques, castrated as adults, were implanted subcutaneously with segments of silastic tubing filled with E2 and with progesterone in a manner such that the levels and the sequence of these hormones mimicked those that occur during the menstrual cycle of intact female macaques. Since previous studies had shown that the difference in cortisol patterns was due to higher levels in females during the day, these studies were conducted from 0800 to 2000 hours. Blood samples were collected in an adjacent room at 15-minute intervals. Separate trials were conducted 2 weeks after E2 was implanted and levels were 110 +/- 14 pg/ml and again 2 weeks later after progesterone was implanted and E2 levels were 59 +/- 15 pg/ml; progesterone levels averaged 4.0 +/- 0.65 ng/ml. Mean plasma concentrations of cortisol (microgram/100 ml) for the 12-hour period were three-fold higher in orchidectomized males treated with E2 (17.2) and with E2 + progesterone (18.0) than in intact males (4.9). Levels in males treated with ovarian steroids were double that (8.5 micrograms/100 ml) observed for intact females.(ABSTRACT TRUNCATED AT 250 WORDS)

Weanling pigs (n = 132) were used to investigate the effects of three common stressors (and a control) and differing social status on behavior, immunity, plasma cortisol, blood chemical, and performance measures. Eleven blocks of 12 pigs each were evaluated. Each pen contained three pigs of dominant (DOM), intermediate (INT), or submissive (SUB) social status. Two weeks later, random pens of pigs experienced either a control treatment (CON) or they were stressed for 4 h by shipping (SHIP), heat-stressed (HEAT) with overhead heat lamps in their home pens, or cold-stressed (COLD) by direct application of water and an air current. Treatments did not influence body weights; however, percentage weight loss during SHIP was greater than for other treatments. Body weights were heavier for DOM pigs than for INT and SUB pigs. Social status had large effects on plasma cortisol, globulin, acute-phase proteins, body weight, and weight changes. Only acute shipping stress resulted in weight loss. Many immune and blood measures were not changed among acutely stressed pigs; however, the relationship between social status and mitogen-induced lymphocyte proliferation and natural killer cell cytotoxicity was disrupted during acute stress. Pig behavior was significantly changed by each stress treatment in a unique manner. During acute stress, behavioral changes seem to be the most consistent and reliable indicators.

Porcine natural killer (NK) cell cytotoxicity, plasma cortisol, total white blood cells (WBC), neutrophil:lymphocyte ratio (N:L), and circulating blood leukocytes were examined from pigs injected i.v. with either saline, ACTH, cortisol, or treated with metyrapone. Plasma cortisol increased (P.05) after ACTH and cortisol treatments and decreased (P.05) after metyrapone treatment; thus, treatments had the intended effects on in vivo cortisol concentrations. In Exp. 1, pigs were injected with either saline or ACTH at 0600 after the initial blood samples were taken (time 0). The ACTH had no effect (P.10) on NK cytotoxicity. Pigs injected i.v. with ACTH had fewer lymphocytes and more neutrophils (P.05) than control pigs. The N:L ratio was greater (P.05) among ACTH-injected than among control pigs. In Exp. 2, pigs were injected i.v. with either saline or 40 or 400 micrograms of cortisol at 0600 after the initial blood samples were obtained (time 0). Cortisol at 40 micrograms had no effect (P.10) on NK cytotoxicity. However, a 400-micrograms bolus of cortisol reduced (P.05) NK cytotoxicity (control = 39.5, cortisol = 28.3% cytotoxicity, SEM = 3.7). Each dose of cortisol reduced (P.05) circulating blood lymphocyte numbers. In Exp. 3, pigs were fed 1 g of metyrapone or no metyrapone the night before sampling. Blood samples were obtained at 0600, 0700, and 0800. Metyrapone reduced (P.05) NK cytotoxicity (control = 28.6, metyrapone = 11.8%, SEM = 1.9). Pigs treated with metyrapone had greater (P.05) numbers of neutrophils than control pigs. Numbers of lymphocytes were greater (P.05) among control than among treated pigs. Pigs treated with metyrapone had a greater (P.05) N:L ratio than control pigs. In conclusion, normal physiological concentrations or moderately increased blood cortisol concentrations did not influence NK activity, although leukocyte distributions were changed. We conclude that greatly increased or greatly decreased circulating concentrations of cortisol reduced NK cytotoxicity.

The secretion of LH, PRL, and cortisol was investigated in 4 sexually mature female rhesus macaques with cardiac catheters protected by tethers. Based on endocrine parameters, all 4 of the animals ovulated within 2 months from the time they were tethered, and regular menstrual cycles of 24-34 days were observed. The catheters remained patent for 6-12 months without reposition or repair. Plasma levels of 2 stress-labile hormones, PRL and cortisol, showed diurnal fluctuations comparable to those observed in untethered animals. The frequency of LH secretory episodes was determined by measuring bioactive LH in blood samples collected at 10-min intervals in the follicular phase and at 15-min intervals in the luteal phase of the menstrual cycle. In 10 trials during the follicular phase, we estimated that an average of between 14 and 15 LH pulses occurred every 12 h. The interpulse interval ranged between 20-80 min and averaged 50 min. No change in pulse frequency was observed across the follicular phase. The number of LH pulses decreased after ovulation, and by the end of the luteal phase, the interpulse interval was 4-6 h. One example during the preovulatory LH surge revealed the high frequency, high amplitude nature of LH secretion at that time. Our experience indicates that tethered animals with cardiac catheters show no hormonal indications of stress and represent the best available model for studies requiring frequent and prolonged access to the vascular system. Our data suggest that peripheral LH fluctuations in rhesus monkeys, as in other mammals, are pulsatile, and the frequency of these pulsatile episodes changes with different phases of the menstrual cycle, presumedly in response to varying stimuli to the pituitary from the brain.

Since male hamsters castrated as adults respond to exogenous estradiol-1 713 (E2) treatment by displaying female-like behavior, we wondered whether males, like females, would release gonadotropin following E2 treatment. To determine this, we performed 2 experiments. In experiment 1, blood samples from adult gonadectomized hamsters of both sexes were collected at 3 h intervals for 48 h starting the day after a midaftemoon injection of estradiol benzoate (EB). Serum levelsof E2, LH and FSH were quantified by radioimmunoassay. Males injected with either 50 or 200 �g of EB showed no LH or FSH surge at any time during the ensuing 2 days. Females, on the other hand, showed midafternoon peaks of LH on both days following the EB injection. In experiment 2,gonadectomized hamsters of both sexes were pretreatedwith E2 -containing implants that elevated serum E2 levels to 170 ± 20 pg/mi (mean ± SEM). The injection of 5Opg ES 1 week later induced a midafternoon surge of both gonadotropins in females but not in males. These experiments show that in hamsters the response of CNS centers that control gonadotropin release to E2 are sexually dimorphic. Furthermore, they suggest that the CNS structures that mediate gonadotropin release in males are more sensitive to masculinizing effects of prenatal androgens than those thatmediate sexual behavior.

In rhesus macaques, the concentration of immunoreactive prolactin in the amniotic fluid remains low during most of the first trimester of pregnancy and then increases abruptly at 60-80 days of gestation. During the second half of pregnancy, large amounts of prolactin accumulate in the amniotic fluid. Much of this amniotic fluid prolactin may originate from the superficial endometrium (decidua). This hypothesis is supported by the increasing amounts of decidual prolactin (dPRL) measured in endometrium obtained at early (50 days), mid-(80 days), and late (greater than or equal to 150 days) gestation. In culture, late pregnancy endometrium released more dPRL than did early pregnancy endometrium. When tissues were cultured in medium without progesterone, the amounts of dPRL measured in the medium declined steadily over 6 days, regardless of the gestational age of the endometrium. dPRL was consistently measured in medium harvested from cultures that received either progesterone or medroxyprogesterone; however, progesterone did not induce an increase in the amounts of dPRL released by cultures prepared from early pregnancy endometrium. This suggests that factors in addition to progesterone may stimulate the increase in dPRL that occurs at midgestation in rhesus macaques.

Elevations in peripheral estradiol (E2) levels inhibit the pulsatile release of pituitary gonadotropins in ovariectomized rhesus monkeys. Efforts to determine the locus of E2 action [i.e. the medial basal hypothalamus (MBH) or the anterior pituitary gland (AP)] have been plagued by the diffusion of crystalline E2 from an implantation site within the MBH to the pituitary portal vasculature and, therefore, to the AP (implantation paradox). With an Alzet osmotic minipump, we delivered 10 ng E2/h to either the third ventricle or the AP of ovariectomized rhesus monkeys for a duration of 18 h, while monitoring changes in the peripheral gonadotropin concentrations. We also infused [3H]E2 into either site to determine the extent of [3H]-E2H]-E2 diffusion by the end of the infusion period. After infusion of [3H]E2 into the third ventricle for 18 h, only trace amounts of [3H]E2 were recovered from the AP. A small amount of [3H]E2 was recovered from the MBH after 18 h of delivery directly to the AP. Infusion of E2 i...

Circadian and ultradian patterns of plasma cortisol were assessed in four intact female rhesus macaques during both the late follicular (days 9-10) and midluteal (days 21-22) phases of the menstrual cycle and compared to patterns in four ovariectomized macaques. Blood samples were drawn from a remote site at 15-min intervals for 24 h via an indwelling catheter. Measures of estrogen and progesterone were obtained for each subject. For purposes of data analyses, group cortisol measurements were collapsed across hourly intervals and submitted to analysis of variance. Pulsatile characteristics of cortisol release were determined for each subject using the PULSAR computer program. Circadian cortisol patterns, present in all three groups, were characterized by a progressive rise during early morning hours (0300-0600 h), followed by a decline of short duration. All groups then displayed an unexpected midday peak (0900-1400 h), at which time cortisol levels reached their daily zenith. In each of the three groups, cortisol levels reached a nadir during late afternoon hours shortly after the light phase ended. The amplitude of circadian changes and daily mean levels of cortisol were significantly reduced by ovariectomy, without alterations in pulsatile characteristics of cortisol secretion. Daily mean cortisol levels decreased from approximately 8 micrograms/100 ml in intact subjects to 4.5 micrograms/100 ml after ovariectomy. No significant differences in the circadian/ultradian periodicity of cortisol secretion were detected between the follicular and luteal groups. When data in the intact female groups were combined and compared to those previously obtained from gonadally intact adult male macaques, similar 24-h patterns of cortisol secretion were detected. Surprisingly, amplitude changes in cortisol concentrations after ovariectomy were temporally and quantitatively similar to those in orchidectomized males. In both male and female animals, circadian patterns of cortisol secretion were reduced by gonadectomy. These results are discussed in terms of the activational influence of gonadal steroids on hypothalamic-pituitary-adrenocortical function.

The hamster uterine luminal epithelium undergoes cyclic morphological changes, which include mitosis and hypertrophy prior to ovulation and degenerative changes after ovulation. We have previously shown that the degenerative changes are symptoms of estradiol (E2) withdrawal; they occur within 24 hours after ovariectomy, can be prevented by E2 treatment, and are associated with a decline in the content of uterine nuclear E2 receptor (West et al., 1978). In this report, the results of an electron microscopic study of the uterine luminal epithelium in cycling and hormonally altered hamsters are described. During estrus and after ovariectomy, the death of numerous epithelial cells occurs by apoptosis. The ultrastructural alterations in such cells include condensation and margination of the chromatin, cytoplasmic condensation, amoeboid changes in cell shape with formation of pseudopodial extensions, and nuclear and cytoplasmic fragmentation. The cell fragments, or apoptotic bodies, accumulate near the basal lamina, and are then phagocytosed by macrophages, and possibly by neutrophils as well. The ultrastructural features of cell death by apoptosis in the hamster uterus are similar to those observed by others in a variety of tissues. Because of the periodic, hormonally regulated occurrence of extensive apoptosis in the hamster uterine epithelium during the estrous cycle, this system is well-suited for further investigations on the ultrastructural alterations that occur in cells undergoing apoptosis. In addition, apoptosis appears to be a sensitive morphological endpoint of E2 withdrawal in the hamster uterine luminal epithelium.

The response of the pituitary-gonadal axis to synthetic LHRH was investigated in fetal (gestational age, 150–159 days), infant (6 months old), and prepubertal (18 months old) rhesus monkeys. In the fetal studies, saline or 80 μg LHRH was adminstered in utero through a cannula placed in the femoral vein. Blood pressure was stable in both fetus and mother throughout each experiment. Immunoreactive serum LH was significantly elevated over control samples in fetal males (n = 3) and females (n = 8) and in infant (n = 6) and prepubertal females (n = 6) after LHRH administration; infant males (n = 4) showed no response. Prepubertal males were not studied. There was no change in LH levels in the maternal circulation after LHRH was given to the fetus, nor was there any change in LH levels in agematched fetuses given saline. Although LH levels were quantitatively different when measured by bioassay and immunoassay, both assays showed an increase in LH after LHRH adminstration. Serum titers of 17β-estradiol (E2) and...

The effects of progesterone on blood levels of FSH and LH during the late luteal and early follicular phase of the cycle were studied in 10 regular cycling rhesus monkeys. Progesterone administered for 8 days in silastic capsules, beginning 12 days after peak concentrations of estrogen in serum, prolonged the luteal phase levels of this hormone in 6 animals. Blood levels of progesterone were not affected in 4 control animals given cholesterol. Throughout two consecutive cycles, progesterone, 20α-hydroxypregn- 4-ene-3-one, estradiol, FSH, and LH were measured by radioimmunoassay at selected intervals. The length of time between the LH peaks in cycle I and II differed significantly (p < 0.05) between animals treated with progesterone (35.8 ± 1.21, SD, days) and those treated with cholesterol (31.0 ± 2.16, SD, days). The concentrations of FSH in the late luteal and early follicular phase of the cycle continued to rise significantly (p < 0.01 by regression analysis) in both treated and control animals in spit...

Serum concentrations of bioactive luteinizing hormone (LH) and of testosterone were measured in adult male rhesus macaques at the time of, and at least 6 wk after orchidectomy. The effects of anesthesia alone and of sham surgery on the secretion of these hormones were also studied. Contrary to expectations based on current notions of the negative feedback actions of testosterone on gonadotropin secretion, pulsatile LH release was absent for an average of 9.3 h immediately after removal of the testes. Animals that were subjected to sham surgery or anesthesia alone showed a delay of 4.4 h in the reappearance of LH secretion, similar to the lull in LH pulsations normally observed at the time of day. When LH secretion resumed in the acutely castrated animals, the pulses continued for the remainder of the sampling period and did not show the midmorning decrease in activity characteristic of intact or sham-operated animals. In the animals that were castrated, circulating cortisol was elevated approximately twofold above levels observed in sham-operated animals for a period of 8 h, beginning immediately after castration. In the long-term (greater than 6 wk) castrated male macaques, circulating bioactive LH levels were eightfold greater (24 micrograms/ml) than levels observed in intact animals (3 micrograms/ml). The frequency of LH pulses in the castrated animals, as determined by PULSAR analysis, was significantly influenced by sampling frequency. When blood samples were taken at 15-min intervals for 24 h, LH pulse frequency averaged 7 pulses/12 h. If samples were collected every 7 min for 12 h, the frequency increased to 13 pulses/12 h.(ABSTRACT TRUNCATED AT 250 WORDS)

In female proestrous hamsters (1800 h), bilateral electrocoagulative lesions in the arcuate-median eminence (Arc-ME) region blocked the pituitary FSH release that normally would have begun in late proestrus and continued until the afternoon of estrus. This second (estrous) FSH elevation, which is not accompanied by LH release, was unaffected by neural disconnection of the medial preoptic area (MPOA) from the medial basal hypothalamus (MBH) or by production of MBH-pituitary islands. After gonadotropin-releasing hormone was injected into Arc-ME-lesioned hamsters, LH was released. These results suggest that the Arc-ME does not require the MPOA for initiation of the estrous release of FSH.

The development of brain stem auditory evoked responses (BAERs), recorded from a surface electrode as short-latency, volume-conducted potentials, was studied in a series of kittens over a postnatal period ranging from birth to 60 days. Repeated, longitudinal observations on particular kittens were supplemented with observations on additional kittens during the first and second postnatal week to determine age of onset of the BAERs. The position of the animal and sound source within the recording chamber were held constant across recording sessions, as was click intensity except during recordings in which intensity effects were specifically studied. Click rates of 1, 10, 50 and 100/sec were routinely presented. Reference electrodes at the tongue, pinna and neck showed volume-conducted responses to the click stimuli and resulted in considerable distortion of the activity recorded by the vertex electrod; the forepaw, in contrast, showed no activity and a vertex-forepaw electrode configuration provided good resolution of the BAERs across development. A number of new observations were made. BAERs were first observed at 4 days of age, approximately the same age at which depth evoked potentials are first recorded in brain stem auditory nuclei. Initially the BAERs were diffuse, high threshold and fatigued rapidly, characteristics shared with depth evoked potentials in the early postnatal period. Over the first two weeks, the potentials showed marked decrease in threshold, increased resistance to fast click rates, and better definition of wave forms. All BAER components showed exponential decreases in latency. Because all of the brain stem evoked potentials could be recorded concurrently and longitudinally in the same subject a number of developmental comparisons were possible among the BAER components. Wave 1, related to the acoustic nerve in the adult cat, showed a developmental time course and adult latency similar to that reported for N 1 . Wave 2, related to the cochlear nucleus in the adult, showed a marked bimodality over the first month; wave 2a was a large amplitude clearly separated wave which gradually fused as an inconspicuous conspicuous leading shoulder on wave 2b. Wave 2b developed with a time course and adult latency similar to that reported for the ventral cochlear nucleus. Wave 3, related to the region of the superior olivary complex in the adult, showed a clear but transient bimodality during the third week of development. Wave 5, related to the inferior colliculus in the adult, appeared later than waves 1–4 and showed a significantly slower rate of development than waves 1–4. These data indicate that differential developmental changes occur within the brain stem auditory pathway and that the BAERs provide a dynamic probe of concurrent maturational interactions.

Peripheral blood samples were taken from 13 rhesus females at 0030, 0630, 0830,1300, and 1600 hr on 4 consecutive days of the follicular (N = 4) and luteal (N = 9) phases of the menstrual cycle and after ovariectomy (N = 4). The first day of menstruation was designated as day 1 and samples were collected on days 5 to 8 and days 19 to 22 of the cycle. Serum levels of progesterone, estradiol and LH were measured by specific radioimmunoassays. None of these 3 hormones showed significant diurnal fluctuations in the follicular phase, but both progesterone and estradiol concentrations varied significantly with time of day in the luteal phase of the cycle. The nadir in circulating progesterone occurred in the morning (0630) and the zenith at night (0030). Estradiol concentration also declined during the morning hours, but less rapidly and out of phase with progesterone. Estradiol was not measured in ovariectomized monkeys, but LH and progesterone concentrations showed similar diurnal patterns. However, ...

Pulsatile secretion of progesterone has been observed during the late luteal phase of the menstrual cycle in the rhesus monkey and human. As the lutealphase progresses in each of these species,there isa pattern of decreased frequency and increased amplitude of progesterone pulses. The present study was designed to determine the pattern of progesterone secretion during the late luteal phase (Days 10-16) of the normal ovine estrous cycle. Five unanesthetized ewes, each bearing an indwelling cannula in the utero-ovarian vein, were bled every 15 mm from 0800 h on Day 10 through 0800 h on Day 16 of the estrous cycle. With the computer program PULSAR, it was determined that progesterone secretion was episodic, with pulsations observed on all days. Analysis of variance was used to determine differences in frequency, amplitude, and interpeak interval (IPI) of progesterone pulses among ewes and days. The ewes averaged 8.0 ± 0.63 pulses of progesterone per 24 h. Mean frequency of pulses was not different between days but showed differences between ewes. Mean amplitude of progesterone pulses was 7.0 ± 0.27 ng/ml, with no differences observed either between days or between ewes. Mean IPI was 197 ± 7.1 mm, and, like frequency, the IPI was not different between days, but varied between ewes. No consistent temporal relationship was found between progesterone pulses and luteinizing hormone (LH), as determined by bioassay and radioimmunoassay, on Day 14 of the cycle in one ewe. The results indicate that progesterone secretion is episodic during the luteal phase of the ovine estrous cycle and is independent of LH pulses. However, a pattern of decreasing frequency and increasing amplitude of progesterone pulses was not observed in the ewe.

Experiments were conducted to examine the pulsatile nature of biologically active luteinizing hormone (LH) and progesterone secretion during the luteal phase of the menstrual cycle in rhesus monkeys. As the luteal phase progressed, the pulse frequency of LH release decreased dramatically from a high of one pulse every 90 min during the early luteal phase to a low of one pulse every 7-8 h during the late luteal phase. As the pulse frequency decreased, there was a corresponding increase in pulse amplitude. During the early luteal phase, progesterone secretion was not episodic and there were increments in LH that were not associated with elevations in progesterone. However, during the mid-late luteal phase, progesterone was secreted in a pulsatile fashion. During the midluteal phase (Days 6-7 post-LH surge), 67% of the LH pulses were associated with progesterone pulses, and by the late luteal phase (Days 10-11 post-LH surge), every LH pulse was accompanied by a dramatic and sustained release of progesterone. During the late luteal phase, when the LH profile was characterized by low-frequency, high-amplitude pulses, progesterone levels often rose from less than 1 ng/ml to greater than 9 ng/ml and returned to baseline within a 3-h period. Thus, a single daily progesterone determination is unlikely to be an accurate indicator of luteal function. These results suggest that the changing pattern of mean LH concentrations during the luteal phase occurs as a result of changes in frequency and amplitude of LH release. These changes in the pulsatile pattern of LH secretion appear to have profound effects on secretion of progesterone by the corpus luteum, especially during the mid-late luteal phase when the patterns of LH concentrations are correlated with those of progesterone.

Six adult ovariectomized rhesus macaques were given a gonadotropin-releasing hormone (GnRH) antagonist [Ac-β-(2)DNAL 1 ,pFDPhe 2 , DTrp 3 , DArg 6 ]-GnRH, by intravenous infusion for 3 to 3.5 days to determine whether the positive feedback action of estradiol benzoate (E 2 B) on pituitary luteinizing hormone (LH) secretion could be inhibited by blockage of GnRH binding to pituitary gonadotropes. The LH release was suppressed when the antagonist was given either as a bolus injection every 6 hours or as a constant infusion, beginning 24 hours after the E 2 B was administered. Both LH release and follicle-stimulating hormone release were suppressed if the GnRH antagonist infusion began when the E 2 B was administered. The results suggest that continued hypothalamic GnRH stimulation of the pituitary is necessary for the full expression of the preovulatory-like gonadotropin surge that occurs in ovariectomized macaques in response to E 2 .

Uteri were removed and blood was drawn from hamsters during the estrous cycle, on the eighth day of pregnancy, and after different hormonal treatments. Serum estradiol (E2) and progesterone (P) levels were determined by RIA. Portions of the uteri were prepared for light and electron microscopy. Endogenous uterine nuclear E2 receptor was measured by exchange assay. Large increments of nuclear E2 receptor occurred when the level of serum E2 was rising and that of serum P was falling (diestrus day 2 through proestrus morning); decrements occurred when the serum E2 level was falling and the serum P level was rising (proestrus evening through diestrus day 1). Ovariectomy on proestrus morning led to a decline in the amount of nuclear E2 receptor, and this decline was prevented by administration of E2 at the time of ovariectomy. P depleted nuclear E2 receptor in the presence of high levels of serum E2. The uterine luminal epithelium passed through a phase of mitosis and hypertrophy (diestrus day 2 through proestrus morning), a brief phase suggestive of secretion (proestrus evening), a phase of degeneration (estrus), and a phase of quiescence (diestrus day I). Ovariectomy on proestrus morning led within 24 h to degenerative changes identical to those seen during estrus, and these changes were prevented by treatment with E2 at the time of ovariectomy. P induced these degenerative changes in the presence of high levels of serum E2. After 8 days of pregnancy, the epithelium resembled that seen during diestrus day 1. Elevated uterine nuclear E2 receptor levels were associated with growth and hypertrophy of the luminal epithelium, and severely depressed levels were associated with the degenerative and quiescent phases of the epithelium.

A study was made of the effects of partial and total deafferentation of the medial basal hypothalamus (MBH) on the estrous cycle, endocrine organ wt, and pituitary and serum LH values in the golden hamster. Complete deafferentation (CC) as well as extended anterior frontal deafferentation (EFC) blocked cyclic ovulation immediately and permanently. Smaller anterior cuts (FC) blocked ovulation for 8–12 days, but this effect was transient, and by 3 weeks following deafferentation the hamsters were showing normal 4–day cycles. Posterior—lateral deafferentation (PL) had no effect, either acute or chronic, on ovulation. Ovarian and uterine wt was drastically reduced in CC animals, indicating a lack of gonadotropin release, and this was confirmed by low serum LH levels and very high pituitary LH concentrations, determined by radioimmunoassay. In EFC animals uterine wt was increased over diestrous and metestrous controls, and their ovaries contained large cystic follicles. Furthermore, serum LH values were elevat...

Progesterone levels were measured at proestrus in intact, phenobarbital (sodium phenobarbital)-treated, hypophysectomized (ℏ) and irradiated hamsters by the competitive protein binding technique. Progesterone content and concentration in the ovary increased dramatically between 2:30 and 3:00 PM and the highest levels were reached at 8 PM. The concentration of follicular progesterone paralleled but was only one half of the total ovarian value. Plasma progestei-one was highest at 6 PM and did not decrease significantly until 12 midnight. Phenobarbital (phen) or ℏ at 1 PM completely blocked ovarian progesterone synthesis at 4 PM. LH but not FSH restored progesterone secretion in ℏ or phen-treated animals. LH significantly increased ovarian progesterone within 15 min in ℏ animals. Hamsters ℏ at 4 PM (after the initial rise in progesterone) continued to show progesterone at control levels for at least 4 hr. Therefore, the continued presence of LH does not appear to be necessary for sustained progesterone secre...

The 00 LH double-antibody RIA system developed by Niswender at al. (1968a) has been tested to show its specificity for LH in the hamster. In this system hamster sera, hamster pituitary homogenates, sheep pituitary LH standard, and rat pituitary LH standard all show parallel dose-response curves. Hamster sera, TSH, and FSH do not appear to interfere with the assay of LII. Reproducibility and precision of the assay are within acceptable limits. The system reveals highly signfficant increases in serum LH after ovariectomy, during the course of proestrus and after in vivo administration of purified LH-releasing hormone. Blood samples were collected at different times up to 1 hr after a single iv, ip, or im injection of synthetic LH-RH. Only iv administration of the releasing hormone caused a rapid rise in circulating LH. At all times up to 1 hr the response seen after an im injection was contained within the dome of the curve representing the response observed after iv administration. Responses seen after ip and im injections were similar.

To determine the acute effect of phenobarbital and progesterone on luteinizing hormone (LH) release and the pituitary adult female hamsters were injected with phenobarbital to block the proestrous release of LH and simultaneously injected with progesterone. After LH-RH administration blood samples were taken for LH study by radioimmunoassay. Partial hypothalamic deafferentation was then performed followed by a 1-mg injection of progesterone just before the proestrous critical period. 1 day later laparotomy was performed and the ovaries examined. A second group of hamsters were ovariectomized and then injected with estrogen and progestagen half of them also receiving phenobarbital and the other half untreated. LH-RH was again administered and blood samples taken. The laboratory tests revealed that phenobarbital did not alter pituitary responsiveness to exogenous LH-RH in either group of animals. The progesterone injection did not change the pituitary sensitivity to the LH-RH in the phenobarbital-treated hamsters indicating that progesterone which normally counteracts a phenobarbital blockade must operate at a neural site. The hypothalamic surgery and subsequent progesterone treatment repeatedly failed to overcome the phenobarbital block. It is concluded that the phenobarbital and progesterone exert their reciprocal actions on the pituitary either outside the hypothalamus or on the synaptic thresholds within the hypothalamus.

Anterior or total deafferentation of the medial basal hypothalamus (MBH) was performed in adult male rats, and LH, FSH, prolactin and testosterone were measured in blood collected by decapitation 6 weeks after surgery. Interruption of the anterior afferents to the MBH significantly reduced circulating levels of prolactin and complete deafferentation resulted in even lower levels. Serum LH, FSH and testosterone were unaffected by the partial deafferentation, but circulating levels of all three hormones were significantly depressed in "complete—cut" rats. Both the concentration and the total content of LH in the pituitary as well as the total pituitary content of prolactin were reduced by complete deafferentation of the MBH. Light microscope examination of the ventral prostate and seminal vesicles in completely deafferented rats indicated a chronic lack of gonadotropic stimulation. The results reveal that the anterior connections to the male MBH do not specifically influence the "tonic" synthesis or release...

Ovarian tissue from adult female rhesus macaques was transplanted into sc abdominal pouches of 4 male rhesus macaques that had been castrated after reaching sexual maturity. The animals were treated daily with cyclosporin A to prevent rejection of the ovarian transplants. Two males in which the transplants were successful showed preovulatory-like gonadotropin surges in response to increasing levels of estradiol. In one of these males (7082), circulating levels of gonadotropins and steroids indicated that cyclic ovarian function had been established. This male showed 5 successive ovarian cycles that averaged 28 days in length. Comparison of the changes in reproductive hormones between 7082 and females with normal menstrual cycles support the hypothesis that the neuroendocrine mechanisms that regulate cyclic release of gonadotropins in primates are not sexually different.

Sexual maturation in female rhesus macaques was studied after surgical isolation of the medial basal hypothalamus [complete hypothalamic disconnection (CHD); n = 4] or after creation of amygdaloid lesions (AMYG; n = 6). In four animals, CHD at 8 months did not affect the age when menarche occurred (30 months) but did result in a significant (P less than 0.01) advancement in age at first ovulation (35.8 +/- 1.1 vs. 43.7 +/- 1.1 months for the five controls). Body weights in CHD and AMYG animals were not different from weights of controls at either menarche or first ovulation, but CHD animals gained weight faster than controls. Although there was no overall difference in age at menarche or first ovulation between AMYG animals and controls, the three AMYG animals that sustained damage to the corticomedial amygdaloid area ovulated later than three other AMYG animals without damage to this area. Daily serum levels of LH, FSH, and 17 beta-estradiol were measured in the second ovulatory cycle of each animal and found to be similar among the three groups. Serum progesterone levels revealed that three of the five controls and one of six AMYG animals had short luteal phases typical of pubertal monkeys, whereas all four CHD animals showed luteal phases typical of sexually mature adult animals. Serum cortisol and PRL showed significant diurnal changes in all three groups. These data indicate that in infant rhesus females, intact neural connections to the medial basal hypothalamus are not obligatory for sexual maturation or for the propagation of entrained diurnal rhythms in cortisol and PRL. That isolation of the medial basal hypothalamus resulted in an increase in the rate of weight gain and favored early sexual maturation may indicate that the main effect of the higher brain centers is inhibitory on hypothalamic mechanisms which control these processes.

Circadian/ultradian patterns of plasma cortisol were assessed in four intact and four gonadectomized male rhesus macaques. Concomitant measures of testosterone (T) were analyzed in the intact animals. Blood samples were drawn every 15 min for 28 h via an indwelling catheter. Plasma concentrations of cortisol and T were determined by RIA. Inverse diurnal patterns of cortisol and T secretion were documented in the control group. The circadian pattern of plasma cortisol was characterized by a progressive rise during the early morning hours (0200-0800 h), followed by a gradual decline until lowest levels were reached at 1600-1900 h. T levels were lowest from 0800-1200 h (2-4 ng/ml) and reached a zenith from 2200-0200 h (8-10 ng/ml). Surprisingly, circadian fluctuations in cortisol were absent after orchidectomy. Cortisol levels in castrates were less (P less than 0.05) than those during peak levels of cortisol secretion in the intact animals (6.25 vs. 10.1 micrograms/100 ml) and elevated (P less than 0.01) compared to concentrations in the intact animals at their nadir (6.4 vs. 2.5 micrograms/100 ml). The loss of circadian fluctuation was not associated with a significant change in frequency of pulsatile cortisol release or a change in the daily mean level. These results are the first in nonhuman primates to demonstrate that the testes modulate circadian activity in the hypothalamic-pituitary-adrenocortical axis. The data differ from previous findings in rodents suggesting that castration increases the synthesis and release of ACTH/corticosterone.